Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria
| Autor: | Sarah Weinecke, Simon J. Moore, Alka Ishwarbhai, Rochelle Aw, Paul S Freemont, James M. MacDonald, Karen M. Polizzi, David W. McClymont, David J. Bell, Kirsten Jensen, Rebekka Biedendieck, Nicolas Kylilis, Argyro Tsipa |
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| Přispěvatelé: | Engineering & Physical Science Research Council (EPSRC) |
| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Transcription Genetic Bacillus Computational biology Biology Ribosome Models Biological cell-free synthetic biology 03 medical and health sciences Synthetic biology Transcription (biology) MD Multidisciplinary Transcription factor Bacillus megaterium automation Multidisciplinary Cell-Free System RNA Promoter modeling Biological Sciences biology.organism_classification Ribosomal binding site 030104 developmental biology PNAS Plus Protein Biosynthesis in vitro transcription–translation Applied Biological Sciences |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America E4349 E4340 |
| ISSN: | 1091-6490 0027-8424 |
| Popis: | Significance Nonmodel bacteria have essential roles to play in the future development of biotechnology by providing new sources of biocatalysts, antibiotics, hosts for bioproduction, and engineered “living therapies.” The characterization of such hosts can be challenging, as many are not tractable to standard molecular biology techniques. This paper presents a rapid and automated methodology for characterizing new DNA parts from a nonmodel bacterium using cell-free transcription–translation. Data analysis was performed with Bayesian parameter inference to provide an understanding of gene-expression dynamics and resource sharing. We suggest that our integrated approach is expandable to a whole range of nonmodel bacteria for the characterization of new DNA parts within a native cell-free background for new biotechnology applications. Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. |
| Databáze: | OpenAIRE |
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